Kidney stones are a common and recurrent disease affecting more than 10% of Americans in their lifetime, but the underlying pathophysiology remains incompletely understood. Since kidney stones occur predominantly during middle age and affect more men than women, US veterans are a high risk group for this disease. Current therapies have limited effectiveness, with only a modest reduction in stone recurrence rates, and there has been no real advancement in preventive therapy for many years. A breakthrough in understanding is clearly needed. Stones form as aggregates of calcium oxalate (CaOx) crystals with layers of organic material, principally urinary proteins, between the individual crystals, and these proteins likely play a critical role in stone formation. The role of proteins in stone formation is obscured by the large number of urinary proteins found within stone matrix. Comparison of urine proteomes from stone forming and normal individuals has failed to identify critical protein differences to date, but our recent Preliminary Data comparing the relative abundances in proteins in CaOx stone matrix to their urinary abundances has highlighted 2 small subsets of proteins that are likely to be key proteins, specifically including both highly anionic and highly cationic proteins. Such a combination of proteins suggests a role for polyanion-polycation aggregates in triggering stone formation, because we have previously shown that such aggregates stimulate CaOx crystal nucleation and aggregation. Therefore, we hypothesize that the relative abundance of proteins critical to the stone forming process will be increased in CaOx stone matrix compared to that seen in freshly voided urine from the same patient, specifically including both highly anionic and cationic proteins. While the proposed experiments will not elucidate the mechanism whereby these specific proteins become enriched in CaOx stone matrix, confirming the presence of this specific protein mixture in a large number of stones is necessary before mechanistic based studies exploring our suggested link between protein aggregation and stone formation are relevant. The proposed work will identify and characterize key proteins in stone formation in two Specific Aims. In Specific Aim 1, we will confirm enrichment of key proteins in stone matrix using quantitative mass spectrometry to determine the relative abundance of proteins in stone matrix and urine samples obtained from 40 CaOx stone forming patients. In Specific Aim 2, the relative abundances of key proteins in urine under stable health conditions will be compared between our stone former panel and a matching normal population to test for the expected increase in key protein abundance in stone former urine. Immunoblot techniques will be used to circumvent problems associated with quantitative mass spectrometry characterization of low abundance proteins. Identification of key proteins associated with CaOx stone formation represents a new paradigm in stone research that can direct future mechanistic studies on protein triggers of stone formation, and hopefully inspire new therapeutic strategies.